Project Summary Streptococcus pneumoniae, also known as the pneumococcus, is a bacterial pathogen that causes a range of human diseases including pneumonia, meningitis and bloodstream infections. This bacterium displays natural competence for genetic transformation and devotes considerable regulatory effort to controlling this process. Although several hypotheses have been advanced, the function of genetic competence is not known. Recent studies in our laboratory suggest that the deleterious mutation rate is particularly high in pneumococcus and lead us to favor the idea that competence serves to help correct deleterious mutations. We propose that, in the absence of repair provided by competence-mediated genetic exchange, pneumococcus will progressively accumulate deleterious mutations resulting in a rapid decline in fitness of the population. The first aim of this project investigates mutation accumulation in S. pneumoniae, as well as the ability of competence to compensate for this process. Evidence for mutation accumulation will be sought using both in vitro and in vivo serial passage models and measuring the fitness of evolved lineages. Mutational differences between ancestral and evolved strains will be determined at the molecular level using microarray-based comparative genome resequencing for a number of isolates. Because the pneumococcus produces high endogenous levels of hydrogen peroxide that appear to promote mutagenesis, the role of oxidative stress in mutation accumulation will also be investigated. The ability of competence to improve the fitness of evolved lineages will be tested in mixing experiments where genetic exchange provides the opportunity to reduce the mutational load. The second aim of this project investigates the role of pneumococcal proteases in the regulation of genetic competence. This aim focuses on the surface protease HtrA that we have shown to repress the development of competence. Experiments in this aim will test the model that digestion of the competence-stimulating peptide by HtrA releases peptide fragments that inhibit further signaling through the competence pathway. Through these two aims, we hope to understand more clearly both the role of competence in the biology of S. pneumoniae and the function of the regulatory pathways that facilitate this role. These studies may provide insight as well into the adaptive strategies of other naturally-competent, pathogenic bacteria that occupy a similar niche colonizing the upper respiratory tract. PUBLIC HEALTH RELEVANCE: Streptococcus pneumoniae, also known as the pneumococcus, is a bacterial pathogen that must first be carried silently in the nasal passages before causing a variety of human diseases including pneumonia, bloodstream infections and meningitis. This project examines how the bacterium avoids progressive genetic breakdown during silent nasal carriage. Understanding this process may lead to new strategies for preventing pneumococcal carriage and disease.